The present invention relates to a method for derivatizing an s-cis-diene compound, a derivatization reagent kit, and a method for analyzing an s-cis-diene compound.
Analysis with a mass spectrometer (hereinafter sometimes referred to as “MS”) is known, for example, in a clinical examination at a hospital. In particular, analysis of a substance derived from a biological body, for example, a hormone with a liquid chromatography mass spectrometer (hereinafter sometimes referred to as “LC/MS”), which involves separating a compound by high performance liquid chromatography (HPLC) and ionizing and analyzing the separated substance with the MS, has high sensitivity and specificity as compared to immunoassay and the like that have hitherto been used. Further, the above-mentioned analysis is capable of performing simultaneous analysis of a number of items. Therefore, the above-mentioned analysis is rapidly becoming used widely in recent years. In quantitative analysis, particularly, with an LC/MS/MS using a tandem mass spectrometer (hereinafter sometimes referred to as “MS/MS”), a plurality of substances can be selectively subjected to quantitative analysis with a selected reaction monitoring (hereinafter sometimes referred to as “SRM”) mode having sensitivity higher than that of LC/MS.
In recent years, as one example of the analysis of a substance derived from a biological body with LC/MS/MS, analysis of vitamin D (hereinafter sometimes referred to as “V.D.”) and vitamin D metabolites in the blood is drawing attention. Vitamin D, which is an s-cis-diene compound, and is a fat-soluble vitamin necessary for regulating calcium metabolism, has an action of increasing the concentration of calcium (Ca2+) in the blood as activated vitamin D (1α,25-dihydroxyvitamin D, hereinafter sometimes referred to as “1,25(OH)2D”). In addition to the above-mentioned action, in vivo metabolites such as 1,25(OH)2D and 25-hydroxyvitamin D (hereinafter sometimes referred to as “25(OH)D”) play important roles in controlling expression of proteins involved in differentiation and growth of cells, production and secretion of a hormone, an immune reaction, and the like. Therefore, vitamin D is classified as a hormone in some cases from the viewpoints of the action mechanism and the function.
As described above, vitamin D and vitamin D metabolites (hereinafter sometimes collectively referred to as “vitamin D”) have bioactivity in a wide range as well as roles as nutrients, and the excess or deficiency of vitamin D is considered to increase the morbidity of various diseases. Therefore, the number of cases of measurement of vitamin D in the blood is increasing and the analysis of vitamin D with LC/MS/MS makes it possible to accurately analyze individual substances having different activities with satisfactory sensitivity than before.
An atmospheric pressure chemical ionization (APCI) method and an electrospray ionization (ESI) method, and the like are used as an ionization method used in LC/MS/MS. The ESI is an ionization method that is most generally used in LC/MS/MS by virtue of the smallest risk of causing fragmentation, a wide range of applicable compounds, and high operability. However, in general, the ESI responsiveness of V.D. metabolites and content of it in the blood is low, the sensitivity may be insufficient even if LC/MS/MS is used for analysis. In view of the foregoing, in order to enhance the detection sensitivity in LC/MS/MS by increasing the ionization efficiency of the vitamin D metabolites, for example, the vitamin D metabolites are analyzed in some cases after being derivatized with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) that is one of Cookson-type derivatization reagents (see, for example, Japanese Patent Application Publication No. 2015-166740). Derivatization of vitamin D with PTAD improves the sensitivity is compared to before derivatization, and enables to perform highly selective detection.
However, further improvement in sensitivity is desired in order to measure a small amount of a sample, for example, blood collected from a newborn baby. For this reason, the inventors of the present invention have developed, as a novel Cookson-type derivatization reagent, 4-(4′-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD) (see, for example, S. Ogawa, et al., Rapid Commun. Mass Spectrom., 25, 2453-2460 (2013)). Vitamin D derivatized with DAPTAD has signal intensity increased by about 100 times that before derivatization, and the derivatization with DAPTAD is suitable for analyzing a small amount of a sample with LC/MS/MS. The signal intensity obtained by the derivatization with DAPTAD is about 10 times that obtained by the derivatization with PTAD that has hitherto been used. Further, derivatizing 25(OH)D, which is one of the vitamin D metabolites with DAPTAD enables to distinguish and quantitate structural isomers such as 3-epimer (3-epi-25(OH)D), which are inactive interference metabolites, and the selectivity is improved than before.
As described above, the derivatization with DAPTAD is suitable for analysis of vitamin D with LC/MS/MS. However, the inventors of the present invention have found that DAPTAD-derivatives of the vitamin D metabolites are partially decomposed during derivatization in some cases due to the influence of an oxidant and the like used for synthesizing DAPTAD. A decomposition product of DAPTAD-derivative of a certain vitamin D metabolite has the same structure as that of a DAPTAD-derivative of another endogenous vitamin D metabolite. Therefore, the decomposition of derivative makes it difficult to perform accurate quantitative analysis of the vitamin D metabolite.